Connexin 43 (Cx43) in the heart is normally expressed at the intercalated disk where it commonly forms junctional channels (gap junctions). Many cardiac diseases produce increased or aberrant expression of Cx43, which results in lateralization of the protein in cardiomyocytes. Our group and others have provided evidence that lateralized Cx43 forms unpaired hyperactive hemichannels (non-junctional channels) at the plasma membrane. Excessive opening of these Cx43 hemichannels is associated with cell death during acute ischemia and cardiac muscular dystrophy. Cell death is thought to result from the loss of electrochemical gradients and small cytoplasmic metabolites through the large and modest selective aqueous pore of connexin hemichannels. We and others have indirect evidence indicating that nitric oxide (NO) opens Cx43 hemichannels, promoting cellular dysfunction in the heart and brain. In addition, we have evidence that under conditions that cause lateralization in cardiomyocytes, Cx43 is S-nitrosated. The molecular mechanisms that cause Cx43 hemichannel opening by NO have not been elucidated, despite the importance this may have in cardiac and other pathologies. We propose that a major contributor to the aberrant opening of undocked hemichannels is Cx43 S-nitrosation. In this proposal, we will take advantage of our expertise in connexin hemichannel biophysics to identify the mechanisms by which NO gates Cx43 hemichannels. Specific Aim 1 will determine whether NO directly opens Cx43 hemichannels via S-nitrosation and identify residues involved. Specific Aim 2 will identify the precise cytoplasmic domain interactions that mediate the effects of NO on Cx43 gating. Specific Aim 3 will determine whether S-nitrosated Cx43 hemichannels promote cell and tissue damage. We plan to accomplish these specific aims using an integrative multidisciplinary approach that combines electrophysiology, mutagenesis, fluorescence and NMR spectroscopy, mass spectrometry, and a cardiac disease model. We expect that our work will contribute to the rational development of new pharmacological strategies to target the specific connexin domains involved in aberrant hemichannel gating. This will allow us to achieve specific therapeutic outcomes, for example, to decrease opening of hyperactive hemichannels during myocardial infarction preventing arrhythmias and tissue damage.